1. Field of the Invention
This invention relates to the recovery of microbially produced chymosin. In particular, this invention is directed to methods for the recovery of chymosin from the fermentation beer arising from culturing microorganisms which have been engineered so as to produce chymosin. This invention further relates to methods for the selective recovery and subsequent purification of microbially produced chymosin.
2. State of the Art
Chymosin is a known enzyme which is particularly useful in the preparation of cheese. Until recently, almost all chymosin employed commercially was recovered from the fourth stomach of a calf, although recovery of chymosin from the stomachs of other mammals, such as lamb, goats, etc. has also been heretofore known. However, due to the recent decrease in calf production, such natural sources of chymosin have declined which, in turn, has provided impetus to microbial generation of chymosin. Thus, recent patents and patent applications have disclosed that chymosin can be produced by the fermentation of genetically engineered microorganisms. For example, the production of chymosin by fermentation of filamentous fungi which have been genetically modified to express and secrete chymosin, is disclosed in U.S. patent application of Lawlis et al., Ser. No. 07/163,219, filed Feb. 26, 1988, which is incorporated in its entirety herein by reference. Likewise, U.S. Pat. No. 4,666,847, which issued on May 19, 1987 and which is incorporated herein by reference in its entirety, discloses the production of chymosin be fermentation of E. coli (a bacteria) which have been genetically modified to express and secrete chymosin as well as the production of chymosin be fermentation of Saccharomyces cerevisiae (a yeast) which has been genetically modified to express and secrete chymosin.
Because the microbial production of chymosin results in the expression of enzymes in addition to chymosin, recovery and purification of chymosin from the fermentation beer has been an ongoing problem. For example, when chymosin is produced in the manner of U.S. Ser. No. 07/163,219, enzymes such as alpha-amylase, acid phosphates, leu amino peptidase, etc., are co-produced during such fermentation. The presence of such additional enzymes in the fermentation beer imparts an additional level of difficulty in the recovery and purification of chymosin from such beers.
While numerous methods are disclosed for isolating enzymes from a fermentation beer, none of the references which Applicants are aware of specifically disclose that chymosin can both be recovered in extremely high quantities, i.e., partition coefficients (K)&gt;85, and selectively from a fermentation beer containing enzymes in addition to chymosin.
For example, U.S. Pat. No. 4,144,130 describes the use of (1) a mixture of a high molecular weight unsubstituted or substituted polyalcohol, polyether, polyvinylpyrrolidone or polysaccharide and an inorganic salt, (2) a mixture of at least two of the above high molecular weight polymers to recover intracellular enzymes from an aqueous solution into which they have been released from the cells. When a mixture of polyethylene glycol and an inorganic salt is used, the desired intracellular enzyme goes into the top polyethylene glycol layer while the cell debris and other fermentation products go into the lower salt-containing layer. This reference discloses that the partition coefficients for various enzymes recovered in the glycol layer was about 0.3 when a normal cell mass was treated, which could be increased to about 3 when frozen cells were mixed with water and disintegrated to release their enzymes. However, this reference does not teach or suggest the selective recovery of a single enzyme, let alone chymosin from a fermentation beer containing more than one enzyme.
Similarly, U.S. Pat. No. 4,728,613, discloses a process for the recovery of extracellularly produced enzymes, such as protease, amylase and microbial rennet, from whole fermentation beer by using an inorganic salt in combination with a polymer selected from the group consisting of polyethylene glycol, an amine derivative of polyethylene glycol, a carboxylate derivative of polyethylene glycol, polypropylene glycol, an amine derivative of polypropylene glycol, a carboxylate derivative of polypropylene glycol, poly(ethylene glycol) ester, polythyleneimine, trimethylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. While the examples of this reference disclose achieving partition coefficients of up to about 80 for such extracellular enzymes, this reference does not specifically disclose the recovery of chymosin from a polyethylene glycol/salt mixture nor does it disclose the selective recovery of chymosin from a fermentation beer containing more than one enzyme.
Likewise, Kula et al., "Purification of Enzymes by Liquid-Liquid Extraction", describes numerous methods for the purification of enzymes by liquid-liquid extraction. Among numerous methods disclosed, Kula et al. disclose that the addition of a polyethylene glycol/inorganic salt mixture to an aqueous solution containing the enzyme will form a two phase system wherein the polyethylene glycol phase will contain the enzyme. Kula et al. further disclose at page 111 that the phase forming polymer (polyethylene glycol) can be removed from the enzyme by adsorption of the enzyme onto ion exchangers; washing away of the phase forming polymer; and the subsequent recovery of the enzyme. However, this reference does not specifically disclose the recovery of chymosin using a polyethylene glycol/salt mixture nor does it disclose the selective recovery of chymosin from a fermentation beer containing more than one enzyme.
On the other hand, U.S. Pat. No. 4,508,825 discloses that extracellular protease and amylase co-produced during the fermentation of a microorganism capable of producing them are separated by the addition of polyethylene glycol and a cationic epihalohydrin/polyamine copolymer or dextran polymer to the fermentation medium and allowing the polymers to phase separate to form a protease rich phase and an amylase rich phase.
Also, U.S. Pat. No. 4,591,563, discloses a process for the simultaneous purification and concentration of the dextran-sucrase enzyme from the culture medium on sucrose. In particular, the disclosed method involves the addition of a polyether such as polyethylene glycol so as to form two phases; the first a heavy dextran-rich phase that contains the concentrated and purified dextran-sucrase enzyme, and the second a lighter polyether-rich phase that contains contaminating enzymatic activities, which is eliminate.
In view of the above, it is apparent that the cited art does not disclose recovery of microbially produced chymosin from a fermentation beer using a two phase liquid-liquid extraction method having partition coefficients for chymosin of greater than about 85.
It is further apparent that the cited art does not disclose the selective recovery of chymosin from a fermentation beer containing fermentation enzymes in addition to chymosin, i.e., most of the chymosin is recovered in one layer whereas most of the other fermentation enzymes are recovered in the other layer.
On the other hand, industrial or commercial scale production of chymosin by microbial activity and its subsequent purification is greatly facilitated by large partition coefficients for chymosin in a liquid-liquid two phase system and also by the selective recovery of chymosin from other fermentation enzymes contained in the beer when such two phase systems are employed.
Accordingly, it is an object of this invention to provide efficient processes for the recovery of microbially produced chymosin from aqueous mixtures of enzymes produced by fermentation or other microbial activity and particularly for commercial scale production of chymosin.
It is a further object of this invention to provide a recovery process for microbially produced chymosin using a liquid-liquid two phase system having partition coefficients for chymosin of greater than about 85.
It is still a further object of this invention to provide a recovery process for microbially produced chymosin using a liquid-liquid two phase system which provides for the selective recovery of chymosin from other polypeptides contained in the fermentation beer including other enzymes.
It is a further object of this invention to provide a process for the recovery and purification of microbially produced chymosin.
These and other objects are achieved by the present invention as evidenced by the attached summary of the invention, detailed description of the invention, examples, and claims.